Plasma levels of triglycerides (TG) and cholesterol have long been recognized as important determinants of cardiovascular disease risk. Among a large number of factors involved, three members of the vascular lipase protein family stand out as critical players in plasma lipid homeostasis. Lipoprotein lipase (LPL) is a principal determinant of plasma TG concentration, whereas hepatic lipase (HL) and endothelial lipase (EL) are primarily involved in the metabolism of HDL-cholesterol. Whereas the regulation of these enzymes by extracellular factors has long been appreciated, post-translational processing within the endoplasmic reticulum (ER) has recently emerged as an important intracellular pathway affecting the expression of active lipases and plasma lipid homeostasis. Using genetic approaches in mouse models, we identified the lipase maturation factor 1 (LMF1) and suppressor/enhancer of Lin-12-like (SEL1L) genes as critical players in the post-translational maturation and secretion of active lipases. Mutations in LMF1 cause combined lipase deficiency and hypertriglyceridemia in mice and humans, whereas a genetic defect in SEL1L leads to impaired LPL secretion from adipose tissue and elevated plasma TG levels in a mouse model. Although these studies implicate LMF1 and SEL1L in lipase biogenesis and lipid homeostasis, the underlying molecular mechanisms remain uncharacterized. In Specific Aim 1, we will characterize the molecular role of LMF1 in the folding and assembly of active lipases within the ER and investigate its interaction with the general chaperone, calnexin (CNX). In Specific Aim 2, we will explore the role of SEL1L in the release of lipases from the ER and investigate functional and structural aspects of a molecular complex between CNX, LMF1 and SEL1L in the coordination of lipase maturation and secretion. In Specific Aim 3, we will investigate LMF1 as a genetic determinant of dyslipidemia in humans through the functional characterization of coding variants recently discovered in hypertriglyceridemic populations. Our studies will contribute to a better understanding of the post-translational processing of vascular lipases, and the molecular mechanisms underlying hyperlipidemia.